Nucleoside monophosphate kinase

Manufactured by Roche

Nucleoside Monophosphate Kinase is a laboratory equipment used to catalyze the transfer of a phosphate group from a nucleotide triphosphate to a nucleoside monophosphate, converting it into a nucleoside diphosphate. It is an essential enzyme involved in the synthesis and metabolism of nucleotides, which are the building blocks of DNA and RNA.

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Lab products found in correlation

Pyruvate kinase, by Merck Group (1 mentions) Vaccinia capping enzyme, by New England Biolabs (1 mentions) Pnk buffer a, by Thermo Fisher Scientific (1 mentions) Alkaline phosphatase, by Roche (1 mentions)

2 protocols using nucleoside monophosphate kinase

Synthesis of Deuterated tRNA and Acetyl-CoA

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To prepare tRNA substrate with a deuterium at C5 of uridine, d⁵-uridine-5′-monophosphate (d⁵-UMP) was synthesized using a protocol for the synthesis of d⁵-uridine^{21 (link)}. In a 1 mL reaction scale, 2.5 mM of d⁵-UMP, 2.5 mM adenosine-5′-triphosphate (ATP), 10 mM phospho(enol)pyruvate (PEP), 0.1 unit Nucleoside Monophosphate Kinase (Roche) and 5 units Pyruvate Kinase (Sigma) were incubated in Kinase Buffer (80 mM Tris-HCl (pH 7.6), 20 mM MgCl₂ and 50 mM KCl) at room temperature for 2 h to produce d⁵-uridine-5′-triphosphate (d⁵-UTP) and the recovered ATP. After reaction, 2.5 mM cytidine-5′-triphosphate (CTP) and 2.5 mM guanosine-5′-triphosphate (GTP) were added, and the mixture was directly used for in vitro transcription of the deuterated tRNA^Arg as described previously.
The deuterated acetyl-CoA (d₃-acetyl-CoA) was synthesized according to a published procedure^{22 (link)}, and d₃-acetyl-CoA was further purified by RP-HPLC.

Selvadurai K., Wang P., Seimetz J, & Huang R.H. (2014). Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism. Nature chemical biology, 10(10), 810-812.

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In Vitro RNA Capping and Labeling

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RNAs were in vitro transcribed and purified as described previously (Lahr et al., 2015 (link)). For studying the un-capped RNAs, transcribed RNAs were treated with alkaline phosphatase (Roche Life Sciences (Indianapolis, IN), cat. no. M183A) and 5’ end-radiolabeled with [γ-³²P]-ATP. To generate capped RNAs, the 5’ triphosphate required for the capping reaction was regenerated in TOP RNAs by incubation of 50 nM RNA with T4 PNK in PNK buffer A (ThermoFisher Scientific cat. no. EK0032) with 3 mM ATP for 20 min at 37°C followed by addition of 5 units of nucleoside monophosphate kinase (Roche Life Sciences, cat. no. 10107948001). RNA was purified by phenol-chloroform extraction, MicroSpin G-25 desalting columns (GE Healthcare Life Sciences (Marlborough, MA), cat. no. 27-5325-01), and ethanol precipitation. RNAs with a 5’ triphosphate were subsequently capped and radiolabeled using vaccinia capping enzyme (NEB (Ipswich, MA), cat. no. M2080S) and [α-³²P]-GTP or GTP according to the manufacturer’s protocol.

Lahr R.M., Fonseca B.D., Ciotti G.E., Al-Ashtal H.A., Jia J.J., Niklaus M.R., Blagden S.P., Alain T, & Berman A.J. (2017). La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs. eLife, 6, e24146.

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